ABSTRACT Alcoholic liver disease (ALD) develops in approximately 20% of alcoholics. The development of ALD is a complex process involving both parenchymal and non-parenchymal cells in the liver, as well as recruitment of immune cells in response to damage and inflammation. Innate immunity and inter-organ cross talk contribute to ethanol- induced liver injury with interactions between intestine and liver of particular importance. Impaired intestinal barrier function is associated with ethanol-induced liver injury in both humans and rodent models. Increased exposure of Kupffer cells, the resident hepatic macrophages, to gut-derived LPS during chronic ethanol activates TLR4-dependent production of inflammatory mediators. Chronic ethanol exposure also sensitizes Kupffer cells to LPS, resulting in increased production of inflammatory mediators. Hyaluronan (HA), an abundant extracellular matrix component, communicates with cells in a size-specific manner. Specific-sized HA fragments are either pro-inflammatory or anti-inflammatory, depending on the HA receptor and cell type involved in the response. We have discovered that specific-sized HA35 (average MW~35kD) normalizes TLR4- mediated signaling in Kupffer cells after chronic ethanol exposure and also protects mice from ethanol- induced gut and liver injury. By Next Generation Sequencing, we have identified a subset of microRNAs that are reciprocally regulated by HA35, providing novel insights into the mechanism of action for both ethanol and HA35 in regulating TLR4 signaling and macrophage polarization Furthermore, our team finds that specific-sized HA35 promotes intestinal health, at least in part by increasing expression of ?-defensins and promoting the formation of tight junctions. Based on the multi-potent functions of HA35, here we will test the hypothesis that HA35 is a dually targeted therapeutic agent in ALD, normalizing Kupffer cell signal transduction after chronic ethanol exposure and protecting the intestinal epithelial barrier. We will address this hypothesis in three Specific Aims. Specific Aim 1: Investigate the mechanism for normalization of TLR4 signaling by HA35 in rat Kupffer cells and human PBMCs. Making use of both bioinformatics and cell based assays, we will 1) investigate the miRNA regulatory pathways reciprocally targeted by ethanol and HA35, which in turn regulate a) the control of nuclear-cytoplasmic shuttling and b) macrophage polarization. Specific Aim 2: Interrogate the impact of HA35 on maintenance of intestinal barrier function. We will use both cultured Caco-2 cells and mouse intestinal organoids to determine mechanisms for the direct effect of HA35 on protecting tight junctions from ethanol. Specific Aim 3: Test the ability of HA35 to prevent and treat chronic ethanol-induced intestinal and liver injury in mice. Importantly, medical grade HA for device-use is commercially available, thus enhancing the likelihood for a rapid translation of our studies on the dually protective functions of HA35 in chronic ethanol exposure into clinical investigations in ALD.